The concomitant danger and global spread of viruses like Ebola, Chikungunya, Enterovirus D68, and Influenza, makes a priority to develop effective vaccines against emerging diseases. In this regard, live vaccines have been effective resources against infections. However, they are deleterious for immune compromised individuals. Thus, there is a critical need for the production of safe virus-free vaccines. For that reason, our goal s to manipulate the mechanisms that regulate the immune responses, in order to generate safe vaccination approaches that could be broadly used by most of the population. In this regard, our preliminary data in collaboration with Dr. Jos Rivera (UPR-Ro Piedras Campus), shows that a novel Supramolecular Nanoparticle (NP) has strong immunomodulator properties when tested in vivo using a DNA vaccination approach. Also, we characterized the immunogenic potential of a Coriolus versicolor polysaccharide (PSP) extract. Our in vitro data shows PSP to stimulate lymphocyte proliferation and to induce a TH1 cytokine profile. This strong preliminary data is very encouraging, and drove our interest to study the immunomodulatory effects of NP and PSP in a DNA vaccination approach. To the best of our knowledge, the immunomodulator activity of any of these novel agents has never been tested in a DNA vaccination regime. We engineered our model antigen to target Dendritic Cells (DCs), as these are potent antigen-presenting cells (APCs). Specifically, HIV-1 Outer Domain-1 (OD1) is a highly-mannosylated motif that routs HIV to Antigen Presenting Cells (APCs). For that reason we tested a plasmid DNA vaccine consisting of a codon-optimized chimera coding for OD1 fused by a linker sequence to the Vaccinia Virus (VACV) A27L antigenic protein to enhance antigen targeting to APCs. Our preliminary data shows that mice immunized with our plasmid (pOD1A27Lopt) produce higher A27-specific cell- and humoral-mediated immune responses, compared to controls. Based on this information, we hypothesize that immune responses produced by pOD1A27Lopt formulated in NP and PSP, will induce long-term immune protection. We will address our hypothesis with the following specific aims: Specific Aim 1. Dose-dependent immunomodulatory effect of our vaccine formulation in cell-mediated immune responses, lymphoproliferation, and cytokine profile. We will compare the effect of NP and PSP in the antigen-specific frequency of CD8+ and CD4+ T-cells releasing IFN-? in spleen cells by ELISPOT analysis, and CD4+ T-cell proliferation. Also, we will study the cytokine profile in serum of immunized mice. Specific Aim 2. Dose-dependent immunomodulatory effect of our vaccine formulation in humoral- mediated immune responses. We will compare the effect of NP and PSP in the antigen-specific production of total IgG, IgG1 (Th2-type), and IgG2a (Th1-type) by ELISA, and the generation of neutralizing antibodies. Specific Aim 3. Immunomodulatory effect of our vaccine formulation in viral replication, memory, activation, and proliferation. We will compare the effect of NP and PSP in the enhancement of antigen-specific protective immunity with a viral challenge after immunization. Also, we will determine the effect of this vaccination protocol on viral replication and induction of long-term memory, activation and proliferation. We expect our vaccine formulation to offer novel approaches for rational-vaccine design against emerging diseases and bioterror agents, advancing the field of immunotherapy. This project will provide new targets for therapeutic interventions to induce safe immune protection.